This invention relates to immunoassays in which the degree of agglutination of reactants is used to indicate the amount of an analyte present in a sample. Agglutination assays have been used for many years in determining the presence or absence of antigens. For example, U.S. Pat. No. 3,171,783 describes the use of agglutination assays for diagnosing pregnancy. More particularly, the invention relates to assays in which agglutination results from the formation of complexes between an antibody for a specific analyte conjugated to a latex particle and another conjugate between the analyte of interest and a carrier molecule. If a sample contains the analyte, it competes with the conjugated analyte and reduces the formation of complexes, thus inhibiting the agglutination. The effect on the degree of agglutination can be measured by absorbance of light in a spectrophotometer. However, the method has disadvantages, for example it is necessary to supply as one of the reagents used in the assay the specific antigen (analyte) which is to be measured. Further, another reagent must contain an antibody specific to the antigen (analyte) to be measured, the antibody also being conjugated to latex particles.
Carrying out agglutination assays requires that an antibody specific to the analyte of interest be obtained, generally by developing such an antibody in an animal and recovering and purifying the antibody for use in an assay. The antibody is conjugated (attached) to latex particles and used as a first component of the assay. The analyte (antigen) which corresponds to the one expected to be in the sample is obtained by a synthetic preparation or by purification from a natural source and then conjugated to a carrier molecule, such as a protein or polymer, and used as a second component of the assay. Since the antigen and the antibody bind together, when the first and second components of the assay are combined, the large complexes described above will be formed. However, when a sample is first mixed with the second component, which contains conjugated analyte, any analyte present in the sample will compete with that in the second component and interfere with the agglutination process. The effect of such interference depends on the amount of the analyte in the sample and it can be determined spectroscopically.
The present invention was discovered unexpectedly, during development of an immunoassay for an analyte (deoxypyridine, Dpd) using the agglutination technique just described. It was found that a non-specific antibody conjugated to latex particles had the ability to combine with an antibody specific to the analyte, resulting in agglutination. This effect was inhibited by the presence of the analyte in the sample and could be measured to determine the amount of the analyte in the sample. It had been expected that agglutination would occur when the analyte conjugated to a carrier combined with the antibody specific to the analyte which had been conjugated to latex particles. However, it was found that the agglutination was occurring even when no analyte was present. Furthermore, the antibody specific to the analyte was binding to a non-specific (generic) antibody, in the absence of the analyte. When the analyte was present in the sample, it interfered with the agglutination in proportion to the amount of the analyte present, making possible a simpler, but similar agglutination assay. As will be seen in the examples below, the new method appears to have general application, since it has been shown to be useable with antibodies from various sources.
Monitoring the presence of pyridinoline and deoxypridinoline has been suggested as a method for determining bone collagen degradation. For example, in U.S. Pat. No. 5,620,861 and U.S. Pat. No. 5,736,344 the amount of an immunocomplex formed between an antibody and pyridinum crosslinks, including the pyridinoline and deoxypridinoline, was measured to indicate the presence of the pyridinum crosslinks. The means used to measure the immunocomplex included the use of a reporter enzyme to produce a colorimetric signal, preferably alkaline phosphatase.
U.S. Pat. No. 4,469,797 discusses a method of monitoring the concentration of digoxin, a drug administered to cardiac patients. It was suggested that various types of immunoassays could be used, including agglutination techniques.